The fungal pathogen Cryptococcus neoformans causes a high incidence of life-threatening infections leading to meningitis in AIDS patients, and this fungus is therefore a major threat to the >40 million people worldwide who are infected with HIV. In addition, the related species C. gattii has recently emerged as threat to immunocompetent people. The infections caused by these fungi are difficult to treat because the pathogens persist in the body, and it is often necessary to maintain patients on life long antifungal therapy to prevent relapse. It is likely that pathogen persistence involves specific adaptations to the host environment that may allow the fungus to withstand the immune response and to resist therapeutic intervention. It is also known that pathogen variation during infection can have a major impact on disease outcome by influencing virulence factor expression, drug resistance and evasion of the immune response. We have discovered a novel mechanism of genomic variation in C. neoformans that may contribute to disease outcome in immunocompromised people. Specifically, we found that a clinical isolate of C. neoformans from an AIDS patient shows copy number variation for chromosome 13 that correlates with variable expression of the virulence factor melanin. That is, loss of melanin production in variants is associated with copy number variation for chromosome 13 and we hypothesize that copy number variants have different virulence properties. Our first aim for the proposed exploratory work is to determine whether copy number variation at chromosome 13 is relevant to the virulence of C. neoformans by testing variants in a mouse model of cryptococcosis. We will also determine whether growth of the pathogen in the host influences the frequency of copy number changes for chromosome 13. Our second aim is to establish a more thorough understanding of copy number variation for all of the chromosomes by examining additional strains from AIDS patients and from environmental sources. We will also use existing samples to examine DNA isolated directly from C. neoformans cells in the cerebral spinal fluid of infected AIDS patients. The results of this work will provide a deeper understanding of C. neoformans variation during infection and contribute to the long- term goal of developing new strategies to combat fungal infections. PUBLIC HEALTH RELEVANCE: The relevance of this project comes from the pressing need to control fungal infections in humans that have impaired immune systems due to AIDS or immunosuppressive therapy. The 40 million or more people infected with HIV are at particular risk of succumbing to fungal disease. The proposed research will specifically examine the ability of a fungal pathogen to vary its genetic make up during the infection process. Pathogen variation may contribute to persistence during infection and treatment failure.